PROJECTS SUPPORTED FOR 2009

Université d’Artois – Lens

The role of ABC transporter in Aβ excretion

SUMMARY OF PROPOSED RESEARCH

Abeta plaques are one important pathological hallmark of AD. Abeta can be degraded by a variety of enzymes including IDE and NEP or excreted via the blood-brain barrier (BBB) by a LRP1/Rage mechanism or more importantly by ABC transporters. Cerebrovascular ABC transporters excrete Abeta from the brain throughout the whole life. Already minor reduction of ABC transporter function leads to continuous accumulation of Abeta within the brain as insoluble intracellular vacuoles, vascular deposits or plaques and soluble Abeta mono- and oligomers. Using ABC transporter deficient mouse models, our previous work shows these drastic effects of intracellular accumulation and exceeded neuronal death in Dutch-type AD mouse model. Unpublished data of subcellular fractionations suggest that animals with reduced ABC transporter function die already at an age of 150d due to the specific accumulation of 6meric and 12meric Abeta. Control animal start with deposition earliest at 2 years of age. Oligomeric Abeta 9mer and 12meres have already been recognized as to be highly toxic to neurons. Moreover, our experiments suggest that the accumulations are also dependent on the type of ABC transporters.

Hypothesis: ABC transporters specifically excrete various Abeta moieties. The reduction of a distinct excretion capacity leads to the accumulation of specific Abeta variants. The kinetics is dependent on the age of the individual and specific ABC transporters. The accumulation of toxic Abeta molecules reduces the number of neurons.

Experimental design: The proposed project will determine the kinetics of Abeta efflux by the use of endothelial cell transwell assays (ECTA) established from single to 4x ABC transporter ko mice available in our lab. Additionally, we will determine the amount of Abeta 40 & 42 and oligomeric moieties in the native mice at different time points throughout their life. Preliminary ELISA measurements at an age of 175 days show that there are huge differences of soluble and insoluble Abeta42 content in different ABC transporter animals expressing APPswe.

Aim: We want to determine to which extent specific ABC transporters contribute to Abeta excretion. Induction of these transporters may supply a new treatment strategy for AD and other proteopathies of the brain.

RELEVANCE OF PROPOSED RESEARCH TO ALZHEIMER’S DISEASE

In recent years, considerable effort has been devoted to the investigation of new drug targets for the treatment of AD. In the field of cancer therapy, the ABC transporters are already recognized to be important for the resistance of cancer cells to pharmaceuticals. We were the first to investigate P-gp, a key ABC transporter involved in the transport of Abeta, in the non-demented elderly. We showed that the amount of Abeta in vessel walls is inversely related to the expression of P-gp. Many drugs are known to influence this putative transporter of Abeta. The detailed investigation of the mechanisms involved will test the rationale for using existing drugs (and for developing new drugs) for AD therapy. Moreover, Ca⁺⁺-antagonists (e.g. verapamil) are widely used to treat hypertension; some of these drugs inhibit ABC transporter function and could therefore enhance Abeta deposition. We think that the investigation and elucidation of these mechanisms is of critical importance to the development of new therapies and assessing the safety of existing pharmaceuticals.

November 1st, 2009 – October 31th, 2011 (2 years)

40 000€

Institut de Pharmacologie Moléculaire et Cellulaire – Valbonne

Implication of nardilysin in Alzheimer’s disease

SUMMARY OF PROPOSED RESEARCH

Alzheimer’s disease (AD), is characterized by the deposition of insoluble proteinaceous aggregates. The culprit protein is the 40-43 amino acid-long amyloid β peptide (Aβ). This fragment is generated from the β-amyloid precursor protein (βAPP) by two distinct enzymes, namely the β- and the -secretases. Beside these Aβ-forming proteases, a third cleavage performed by the so-called -secretase takes place in the middle of the Aβ sequence and not only precludes its formation but also generates the secreted product sAPP that possesses neurotrophic and neuroprotective properties. This beneficial cleavage has been shown to be triggered directly or indirectly by proteases of the ADAM family (A Disintegrin And Metalloprotease). Thus, ADAM10 and ADAM17 are respectively responsible for constitutive and protein kinase C-dependent -secretase cleavage of βAPP.

It has been shown very recently that the N-arginine dibasic convertase nardilysin (NRDc) was able to activate ADAM17 and promote the -secretase cleavage of βAPP as demonstrated by the strong increase in sAPP release and the concomitant reduction of Aβ secretion. Unexpectedly, the catalytic site of NRDc is not required for ADAM17 activation whereas physical interaction between the two proteins seems to be necessary.

The objectives of the present research project could be divided in two main parts: the first aspect concerns the detailed impact of NRDc overexpression/invalidation on the -secretase metabolism of βAPP and the degradation of Aβ. One priority will be to determine 1) whether NRDc is able to degrade one or several Aβ species (secreted and intracellular Aβ40, Aβ42) and to engender N-terminal truncated forms of Aβ that are known to be highly toxic and 2) whether NRDc can affect apoptosis through its action on Aβ. For that purpose we will transfect NRDc in different cell lines including primary neurons prepared from mouse models of Alzheimer’s disease and quantify Aβ species by immunoprecipitation with specific antibodies. The second part of the project will focus on the yet unknown mechanisms by which NRDc activates ADAM proteases. By means of NRDc transfection in cells followed by sucrose gradient fractionation or phosphorylation assays, we will investigate the effect of NRDc overexpression/invalidation on the subcellular localization of ADAM proteases and on its ability to facilitate ADAM17 phosphorylation/activation by kinases.

RELEVANCE OF PROPOSED RESEARCH TO ALZHEIMER’S DISEASE

Alzheimer’s disease, as well as most of other neurodegenerative disorders, is characterized by the deposition of insoluble forms of the amyloid β peptide (Aβ). Hence, any intervention aimed at reducing its production and/or accelerate its degradation could be envisioned as therapeutic ways to slow down the disease. Interestingly, the -secretase metabolism of βAPP not only occurs in the middle of the Aβ sequence, thus preventing its production, but also gives rise to the production of the large soluble neurotrophic and neuroprotective sAPP fragments. In this context, the very recent identification of nardilysin as an indirect activator of the -secretase cleavage, together with the fact that this enzyme also belongs to the same family as the Aβ-degrading enzyme insulinase, makes this protein a very promising candidate regarding new therapeutic perspectives for the treatment of Alzheimer’s disease.

November 1st, 2009 – October 31th, 2011 (2 years)

75 000€

CNRS, Université Pierre et Marie Curie – Paris

Can Aβ explain all PS1 effects on the synapse?

SUMMARY OF PROPOSED RESEARCH

Alzheimer’s disease (AD) is a senile dementia which histopathological hallmarks are the amyloid plaques (aggregated amyloid peptide) and tangles (aggregated hyperphosphorylated Tau).It is well established that cognitive deficits correlate better with synaptic dysfunction than to amyloid load. Therefore understanding early synaptic dysfunction may help to cure AD before dementia. Presenilin 1 (PS1) mutations are responsible for the majority of familial AD. The amyloid hypothesis predicts that synaptic dysfunction, amyloid plaques, tangles and cell death are caused by an excess of β-amyloid (β).

We chose to investigate synaptic plasticity starting from PS1(M146V) Knock-in mice which exhibit deficit in learning dependent spatial tasks. Previous studiews have shown that early LTP – the cellular correlate of memory – is paradoxally increased in PS1(M146V)KI mice. Aβ which is hypersecreted in PS1 mutated mice should have reduced the early LTP: it is thus possible the phenotype observed is not solely due to Aβ However PS1(M146V)KI increases the release of rodent Aβ which in contrast to human Aβ does not aggregate and only aggregated human Aβ is toxic. We think that it would be interesting to study LTP in the CA1 region of hippocampal slices, amyloid load and Tau hyperphosphorylation in PS1(M146V)KI mice which hypersecrete human Aβ. To do this we will cross wild type human amyloid precursor protein (APP) mice with PS1(M146V)KI mice in order to obtain double transgenic mice APP/PS1(M146V)KI mice. This part of the study will be performed in 3,6,12 month old mice. PS1 is part of the -secretase complex which processes APP but also other type I membrane proteins. So we wish to investigate the Aβ independent effect of PS1(M146V)KI. To do so we will investigate early and late LTP in double transgenic mice APP Knock-out/PS1(M146V)KI in CA1 region of hippocampal slices in 3, 6 12 month old mice. At these different ages we will perform Western blots of Tau to detect hyperphosphorylated Tau. We will also perform immuhistochemical detection of Tau inclusion. In order to get an insight on the molecular pathway involved in the increase of the early LTP and decrease in the late-LTP (preliminary results) slices will be frozen at the end of the recording for future Western blotting. Among the pathway analysed we will focus on the netrin-1 receptor of DCC which is intimately linked to PKA which plays a role in the LTP. Another pathway that will be analysed is the PI3-kinase pathway which was shown to be modified by PS1 mutation. Overall this study should shed light on the mechanism of PS1(M146V)KI effects on synaptic dysfunction.

RELEVANCE OF PROPOSED RESEARCH TO ALZHEIMER’S DISEASE

Understanding the mechanisms involved in the effect of PS1 mutation should help to understand this type of familial AD. Also understanding PS1 function is also understanding some mechanism involved in sporadic AD since the gene of PS1 is upregulated in these conditions. This study should shed light on the respective roles of Tau and Aβ in AD disease model.

November 1st, 2009 – October 31th, 2011 (2 years)

75 000€

NSERM, Institut Pasteur – Lille

Research of functional polymorphisms in Alzheimer’s Disease

SUMMARY OF PROPOSED RESEARCH

Late-onset Alzheimer Disease (AD) is a multifactorial neurodegenerative disease. If the AD risk is in a large part attributable to genetic factors, only the 4 allele of the apolipoprotéine E (ApoE) gene has been characterized as a major determinant of the AD. Numerous association studies were performed in order to identify new genetic determinants of the AD, but very few genes are consistently associated with the AD, and they are all minor determinants. Today, the study of AD genetics turns toward high-or very high-throughput genotyping analyses in very large populations in order to have sufficient statistical power to characterize the polymorphisms the genes involved.

Our laboratory conducted a two stage genomic-wide association study. Firstly, we observed 12 loci exhibiting suggestive and independent associations with the association with the risk of developing AD in a French population (2,032 cases and 5,328 controls). Secondly, the association of 21 Tag-SNPs within these loci was tested in 3,272 cases and 2,514 controls obtained from Italy, the Netherlands and Spain. Currently, we have analyzed 16 Tag-SNPs in 10 locis and we have observed that 8 of them in 4 loci are associated with AD risk (P<0.05). Two of these loci have reached genome-wide significant association level when all individuals (stage 1 + stage 2) were analyzed together.

Our objective is to identify the functional SNPs explaining the association of these two genes with the disease and to characterize how they contribute to modulate the risk of AD.

First, we will characterize all the SNPs within the gene of interest in all the AD cases and controls DNA sample from a population originated from Lille : after enrichment of the sequences of interest (NimbleGen Technology) in two pools of samples according to their status, the 2 genes will be completely sequenced (by high-throughput sequencing) (i) to establish an exhaustive list of all the SNPs within these genes, (ii) to measure the average allele frequency of each SNP, (iii) to estimate the number of allele of each SNP and (iv) to estimate the association of all minor allele with the risk of developing AD in the two pools of DNA sample.

Second, the SNPs associated with AD risk at an allelic level, will be individually genotyped in all the populations available in the laboratory (using a BeadXpress Illumina station) in order to validate the initial association, to determine the genetic model involved and to perform adjustment for covariables (age, gender, APOE status). The candidate functional SNPs will be restrained according to the result of these association studies, haplotypes analyses and their potential impact on the protein expression or function.

Biological models will be then accordingly developed to characterize the SNPs functionality, such as enzymatic assay, protein quantification, immunochemistry experiment; DNA-protein binding, and transcriptional activity studies, mRNA stability, alternative splicing studies or mRNA translation studies.

RELEVANCE OF PROPOSED RESEARCH TO ALZHEIMER’S DISEASE

This project is designed for a better understanding of the pathological process of AD by the investigation of its genetic determinants. These genetic factors could be used as new pharmacological targets or be useful for diagnosis.

November 1st, 2009 – October 31th, 2011 (2 years)

75 000€